Enterococcus faecalis and Enterococcus faecium are gram-positive bacteria that commensally colonize the lower intestinal tract, oral cavity and vaginal tract of humans. In healthy individuals, E. faecalis and E. faecium colonization normally has no adverse effect on the host. However, the incidence of enterococcal infections has increased and since the 1970s and 1980s, enterococci have become major nosocomial pathogens. Enterococcal infections are mostly hospital-acquired though they are also associated with some community-acquired infections. Recent evidence suggests that E. faecium can be spread by direct contact with other infected patients, indirect transmission from hospital personnel (J M Boyce et al., 1994, J Clin Microbiol 32:1148; and E. Rhineheart et al., 1990, N Engl J Med 323:1814), or from contaminated hospital surfaces and equipment (L V Karanfil et al., 1992, Infect Control Hosp Epidemiol 13:195; and J M Boyce et al., 1994, J Clin Microbiol 32:1148; and L L Livomese Jr., 1992, Ann Intern Med 117:112). Of nosocomial infections enterococci account for 12% of bacteremia, 15% of surgical wound infections, 14% of urinary tract infections, and 5 to 15% of endocarditis cases (Huycke, M. M., D. F., Sahm and M. S. Gilmore. 1998. Emerging Infectious Diseases 4:239-249). Additionally enterococci are frequently associated with intraabdominal and pelvic infections. They are now the fourth leading cause of hospital-acquired infection and the third leading cause of bacteremia in the United States. Fatality ratios for enterococcal bactermia range from 12% to 68%, with death due to enterococcal sepsis in 4 to 50% of these cases. See T. G. Emori (1993) Clin. Microbiol. Rev. 6:428-442. Enterococcus faecalis or E. faecalis is the most common pathogen in the group of these pathogens, accounting for 80-90 percent of all enterococcal infections. See Lewis et al. (1990) Eur J. Clin Microbiol Infect Dis. 9:111-117.
Enterococcal infection is of particular concem because of the emergence of bacteria that are resistant to antibiotics such as aminoglycosides, penicillin, ampicillin and vancomycin. The acquisition of virulence factors and high-level antibiotic resistance by enterococci are causing these organisms to emerge as a leading source of nosocomial infections, particularly in immunocompromised patients. The development of multiple-drug resistant (MDR) enterococci has made enterococcal bacteria a major concern. For example, E. faecium is a prominent cause of infection in humans, with high levels of multidrug resistance (A. Kaufhold and R. Klein (1995) Zentralblatt fuer Bakteriologie 282 507). Antimicrobial resistance can be divided into two general types, 1) inherent or intrinsic resistance and 2) acquired resistance. The genes for intrinsic resistance, like other species characteristics, appear to reside on the chromosome. Acquired resistance results from either a mutation in the existing DNA or acquisition of new DNA. The various inherent traits expressed by enterococci include resistance to semisynthetic penicillinase-resistant penicillins, cephalosporins, low levels of aminoglycosides, and low levels of clindamycin. Examples of acquired resistance include resistance to chloramphenicol, erythromycin, high levels of clindamycin, tetracycline, high levels of aminoglycosides, penicillin by means of penicillinase, fluoroquinolones, and vancomycin. Resistance to high levels of penicillin without penicillinase and resistance to fluoroquinolones are not known to be plasmid or transposon mediated and presumably are due to mutation(s).
One promising approach to the detection and treatment of pathogenic gram positive bacteria is the use of bacteriophage lytic enzymes as bacteriolytic agents. Bacteriophage lytic enzymes responsible for bacterial host lysis are also known as lysins. Many lysins can rapidly break down the bacterial cell wall in order to release progeny phage (Young, R. 1992. Bacteriophage lysis: mechanism and regulation. Microbiol. Rev. 56:430-481). Structurally, lysins are commonly found as modular proteins with an amino terminal domain that confers the enzymatic activity for a peptidoglycan bond and a carboxy terminal domain that confers binding specificity to a carbohydrate epitope in the bacterial cell wall (Loessner, M., K. Kramer, F. Ebel, and S. Scherer. 2002). C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates. (Mol. Microbiol. 44:335-349; Lopez, R., E. Garcia, P. Garcia, and J. L. Garcia. 1997). The pneumococcal cell wall degrading enzymes: a modular design to create new lysins? (MicroB. Drug Resist. 3:199-211; Lopez, R., M. P. Gonzalez, E. Garcia, J. L. Garcia, and P. Garcia. 2000). Biological roles of two new murein hydrolases of Streptococcus pneumoniae representing examples of module shuffling. (Res. Microbiol. 151:437-443; Sheehan, M. M., J. L. Garcia, R. Lopez, and P. Garcia. 1997). The lytic enzyme of the pnemococcal phage Dp-1: a chimeric enzyme of intergeneric origin. (Mol. Microbiol. 25:717-725). Lysins are believed to provide at least one of the following enzymatic activities against a peptidoglycan substrate: muramidases, glucosaminidases, N-acetylmuramyl-L-alanine amidase and endopeptidases (Young, R. 1992. Bacteriophage lysis: mechanism and regulation. Microbiol. Rev. 56:430-481). Purified lysin from a bacteriophage can be applied exogenously to affect bacterial lysis (Loeffler, J. M., D. Nelson, and V. A. Fischetti. 2001. Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science. 294:2170-2172; Loessner, M., G. Wendlinger, and S. Scherer. 1995). Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes. (Mol. Microbiol. 16:1231-1241; Loessner, M., S. K. Maier, H. Daubek-Puza, G. Wendlinger, and S. Scherer. 1997). Three Bacillus cereus bacteriophage endolysins are unrelated but reveal high homology to cell wall hydrolases from different bacilli. (J. Bacteriol. 179:2845-2851; Nelson, D., L. Loomis, and V. A. Fischetti. 2001). Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Prot. Natl. Acad. Sci. USA. 98:4107-4112).
Lysins are normally very specific to the bacterial species from which the lysin derived phage was isolated (Fischetti, V. A. 2003. Novel method to control pathogenic bacteria on human mucous membranes. Ann. N.Y. Acad. Sci. 987:207-214; Fischetti, V. A. 2001. Phage antibacterials make a comeback. Nature Biotechnol. 19:734-735). Although the range of bacteria targeted by lysins is less restrictive than the corresponding bacteriophage, lysins still maintain a degree of specificity, having minimal effects on other bacteria including commensal organisms. While bacteriophage host ranges are largely restrictive, recognizing only one specific antigen on its bacterial host, phage lysins are less restrictive, recognizing a specific carbohydrate molecule common to the particular species of host bacteria.
Bacterial resistance to phage lysins is believed to be less likely to arise as compared with bacteriophage adsorption. Bacterial lysis upon exposure to lysin is almost immediate, not giving bacteria much possibility for mutation and secondly, because lysins bind to highly conserved molecules in the bacterial cell wall that are under selective pressure not to mutate. This is evidenced by the lysins from S. pneumoniae phages binding to choline, an essential component on the S. pneumoniae cell wall, and a lysin, PlyC, targeting S. pyogenes by specifically binding the alternating (α1→2) and (α1→3) linked polyrhamnose backbone of surface carbohydrates. The exposure of bacteria to subinhibitory lysin concentrations and mutagenesis studies have not identified bacteria that are resistant to the action of phage lysins (Schuch, R., D. Nelson, and V. A. Fischetti. 2002. A bacteriolytic agent that detects and kills Bacillus anthracis,” Nature, 418:884-888). In contrast, bacterial resistance to many antibiotics are easily identified using the techniques used above. Furthermore, the problem with lysogenic conversion is completely eliminated with phage lysins, and animal testing have determined lysins to be safe.
There is an ongoing need for therapies and agents effective in the diagnosis and control of bacterial contamination, colonization and infection. In addition, compounds with bacteriocidal effects may be useful in the decontamination of bacteria on inanimate surfaces and objects. The bactiophage lytic enzymes provided herein are useful in providing agents useful in the detection, treatment and decontamination of gram-positive bacteria, including Enterococci bacteria.